Protein phosphorylation is an important mechanism for post-translational regulation of glutamate receptors. Through phosphorylating a specific amino acid in the intracellular domain, protein kinases regulate anchoring, trafficking, and signaling of a given glutamate receptor. Group I metabotropic glutamate receptors (mGluR1/5) are densely expressed in the striatum, a brain area involved in addictive properties of psychostimlants. The long-form mGluR1/5 splice variants (1a, 5a, and 5b) have a large intracellular C-terminal tail, which provides a basis for direct protein-protein interactions and phosphorylation. In our recent studies, we found that Ca2+/calmodulin-dependent protein kinase II (CaMKII) binds directly to the proximal region of mGluR5a C-terminus. This binding converts mGluR5a into a biochemical substrate for phosphorylation likely at a selective serine site. These findings raise innovative questions as to if CaMKII regulates mGluR1/5 via a direct protein-protein interaction and phosphorylation and if this regulation has a high clinical relevance in a disease model. In this continuation proposal, a series of coherent experiments from molecule to behavior was proposed to confirm the direct binding of CaMKII to mGluR1/5 in vitro and to establish that native CaMKII and mGluR1/5 interact with each other in striatal neurons in vivo. We will characterize if and how Ca2+ regulates the interaction between CaMKII and mGluR1/5 in vitro and in vivo. We will then investigate whether Ca2+-regulated CaMKII-mGluR1/5 interactions regulate 1) signaling efficacy of mGluR1/5, 2) trafficking of the receptors, and 3) interactions of mGluR1/5 with key scaffold Homer proteins, in striatal neurons or heterologous cells. Finally, we will carry out neurobehavioral experiments to define the role of CaMKII-mGluR1/5 interactions in the addictive action of the psychostimulant amphetamine. Our results will provide evidence and insights for a new synaptic model of kinase-regulated mGluRs and for its linkage to a mental illness (substance addiction). They will also ultimately contribute to the development of novel pharmacotherapies, by targeting mGluRs and CaMKII, for treating various mental illnesses, including addiction. PUBLIC HEALTH RELEVANCE: This research project is aimed to elucidate molecular mechanisms underlying the regulation of metabotropic glutamate receptors and roles of the receptor in drugs of abuse. The information obtained through this project is valuable for the development of new pharmacotherapies for mental illnesses stemming from dysfunctional glutamatergic transmission in the central nervous system.